Pyruvate kinase: Function, regulation and role in cancer.

Serine metabolism is frequently dysregulated in cancers; however, the benefit that this confers to tumors remains controversial. In many cases, extracellular serine alone is sufficient to support cancer cell proliferation, whereas some cancer cells increase serine synthesis from glucose and require de novo serine synthesis even in the presence of abundant extracellular serine. Recent studies cast new light on the role of serine metabolism in cancer, suggesting that active serine synthesis might be required to facilitate amino acid transport, nucleotide synthesis, folate metabolism, and redox homeostasis in a manner that impacts cancer.

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The importance of serine metabolism in cancer.

PKM2 contributes to cancer metabolism.

Aerobic glycolysis is the dominant metabolic pathway utilized by cancer cells, owing to its ability to divert glucose metabolites from ATP production towards the synthesis of cellular building blocks (nucleotides, amino acids, and lipids) to meet the demands of proliferation. The M2 isoform of pyruvate kinase (PKM2) catalyzes the final and also a rate-limiting reaction in the glycolytic pathway. In the PK family, PKM2 is subjected to a complex regulation by both oncogenes and tumour suppressors, which allows for a fine-tone regulation of PKM2 activity. The less active form of PKM2 drives glucose through the route of aerobic glycolysis, while active PKM2 directs glucose towards oxidative metabolism. Additionally, PKM2 possesses protein tyrosine kinase activity and plays a role in modulating gene expression and thereby contributing to tumorigenesis. We will discuss our current understanding of PKM2's regulation and its many contributions to tumorigenesis.

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PKM2, a Central Point of Regulation in Cancer Metabolism

The major nitrogenous osmolytes present in the cells of marine invertebrates, notably the free amino acids glycine, alanine and proline, and trimethylamine oxide and betaine, are highly compatible with proper enzyme function and structure. These nitrogenous osmolytes display either non-perturbing or, in some cases, favorable effects on enzyme-substrate and enzyme-cofactor complex formation, catalytic velocity and protein structural stability. In contrast, inorganic salts (KCl and NaCl) and certain of the free amino acids which play only a minor osmotic role, e.g., arginine and lysine, have strongly perturbing effects on one or more of these enzymic parameters. The compatible nitrogenous solutes therefore are suitable for use at high (several tenths molar) concentrations and at widely varying concentrations in osmo-conforming species. Certain nitrogenous solutes, especially trimethylamine oxide, betaine and glutamate, offset some of the perturbing effects of inorganic ions on enzyme function. The selective accumulation of osmolytes thus involves not only the concentration of non-perturbing solutes, but also a balanced accumulation of solutes with opposing effects on enzymes. The selection of end-products of anaerobic metabolism also appears to be based, in part, on considerations of solute compatibility with enzyme function. Octopine is a non-perturbing solute, whereas arginine, which is condensed with pyruvate to form octopine, is very strongly perturbing. Succinate has marked stabilizing effects on protein structure. We conclude that the composition of the intracellular fluids of marine invertebrates reflects selection for osmolytes and end-products whose net effects create a cellular microenvironment which is conducive to optimal enzyme function and structure. The accumulation of compatible solutes may preclude the necessity for widespread changes in protein structure in adapting to concentrated or highly variable osmotic environments.

Solute compatibility with enzyme function and structure: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates.

Alanine, serine, and phenylalanine behave as inhibitors competitive with phosphoenolpyruvate for the activated forms of the chicken pyruvate kinases. On the other hand, phenylalanine and alanine behave as K-type inhibitors and serine behaves as a heterotropic activator of pyruvate kinase variants which undergo homotropic activation. Tryptophan lowers the Vm and tends to yield complex plots with all variants studied. Kinetic patterns obtained in the presence of phenylalanine also show some characteristics not generally associated with a competitive mechanism. These observations are related to data previously obtained using the rat isozymes and are used to formulate a mechanism which explains the effects of the amino acids. This mechanism hypothesizes that all the effector amino acids bind to the phosphoenolpyruvate site; however, amino acids with nonpolar side chains also interact with a nonpolar region of the T conformer and thereby stabilize it. It is further proposed that there are two such nonpolar regions on the various pyruvate kinases--the one which reacts with the nonbulky side chains, and another which reacts only with relatively bulky side chains. The stabilizing effect of this second nonpolar interaction imparts inhibitory characteristics which are not competitive in nature. Serine and perhaps other polar compounds may also bind at the phosphoenolpyruvate site, but because of their polarity exert a repulsive force at the same nonpolar site with which the nonbulky nonpolar amino acids interact. This repulsion stabilizes the R conformation. Presumably the homotropic activating effects of phosphoenolpyruvate operate via this same mechanism. The data are also used to support a specific sequential-concerted mechanism for the homotropic activating effect of phosphoenolpyruvate. According to this mechanism, phosphoenolpyruvate adds sequentially to the first two subunits. This interaction causes the respective subunits to convert to the R conformation but, once two subunits are in R conformation, the remaining two subunits convert in concert.

Inhibition of chicken pyruvate kinases by amino acids.

Chicken embryos less than 15 days old contain only the K isozyme of pyruvate kinase, which appears to exist in vivo as an R,T conformational set with pI values of 7.2 and 6.6, respectively. Sets of lower pI and higher pI K-isozyme variants also are obtained. Whole embryos of 15 days or more of development show progressively increasing amounts of higher pI, lower K0.5S enzymatic variants. Tissue distribution and kinetic properties suggest that the highest pI form (pH 8.8-9.0) is an M-isozyme analogue. The intermediate forms are postulated to be hybrids. Adult liver extracts contain only the embryonic K isozyme; no evidence for an L-isozyme analogue was obtained. All major forms of the enzymes are compared with respect to saturation by phosphoenolpyruvate in the absence of effector and in the presence of fructose 1,6-diphosphate, alanine, serine, phenylalanine, tryptophan, and/or Mg-ATP.

Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinases.

1. 1. The pyruvate kinase (EC 2.7.1.40) isozyme composition of adult and developing Xenopus laevis tissues was investigated using isoelectrofocusing, DEAE-chromatographic, heat stability and kinetic studies. 2. 2. A total of nine different pI variants were identified. Evidence was presented supporting the concept that the lowest pI form (pH 5.0) was the L-isozyme, an intermediate pI form ( pH = 6.0) was the K-isozyme and the highest pI form ( pH = 7.0) the M-isozyme. The remaining forms were suspected KL and KM hybrids having pI values intermediate to the respective homotetrameric forms. 3. 3. Adduced evidence showed that in vitro incubation could convert the K-isozyme to forms having pI values of suspected KM hybrids indicating possible conversion of K- to M-type subunits. 4. 4. Skeletal muscle is characterized by expression of M-subunits at a high specific activity; heart muscle also has a relatively high specific activity which, however, is primarily due to K-type subunits. 5. 5. The developing embryo initially expresses both K and L type subunits but the specific activity decreases rapidly with development. By the neural crest stage, the expression of the L-subunit is essentially nil and some M-type subunits start to appear. It is suggested that the initial expression of K- and L-subunits is due to the presence of long-lived maternal m -RNA.

Steven W. Marles

Papers

Inhibition of chicken pyruvate kinases by amino acids.

Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinases.

Distribution of pyruvate kinase isozymes in adult and developing Xenopus laevis

An isoelectrofocusing study of rabbit pyruvate kinases